Derivatives of n-2-hydroxyphenylthiourea

ABSTRACT

N-2-hydroxyphenylthiourea derivatives are disclosed, possessing antiviral activity against foot and month disease in mice and guinea pigs and Coxsackie A and B infections in newborn mice.

United States Patent [191 Shindarov et a].

[111 3,846,491 1 Nov. 5, 1974 DERIVATIVES OF N-2-HYDROXYPHENYLTHIOUREABulgaria [73] Assignee: Medizinska Akademia, Sofia,

Bulgaria [22] Filed: Aug. 23, 1972 [21] Appl. No.: 282,917

[30] Foreign Application Priority Data Aug. 23, 1971 Bulgaria 17223 [52]U.S. Cl 260/552 R, 260/553 A, 424/322 [51] Int. Cl. C07c 157/08 [58]Field of Search 260/552 R, 553 A [56] References Cited UNITED STATESPATENTS 2,795,610 6/1957 Gerjovich 260/553 A OTHER PUBLICATIONS Simov,Chemical Abstracts, Vol. 74, p. 438, (item 99167x), (1971).

Herrmann, Proc. Soc. Exp. Biol., Vol. 107, pp. 142-145, (1961).

Primary ExaminerLeon Zitver Assistant ExaminerMichael W. Glynn Attorney,Agent, or Firm-Armstrong, Nikaido & Wegner [57] ABSTRACTN-Z-hydroxyphenylthiourea derivatives are disclosed, possessingantiviral activity against foot and month disease in mice and guineapigs and Coxsackie A and B infections in newborn mice.

2 Claims, No Drawings BACKGROUND OF THE INVENTION In view of the factthat treatment of viral infection diseases represents a serious problemin modern medicine, research work for discovering efficient preparationsfor ethiotropic treatment having inhibiting effect on the virus growthis of great importance for modern virology.

Among the numerous compounds studied in the prior art, only some areknown to possess antiviral activity. Izatine-B-thiosernicarbazonederivatives have been proposed for smallpox prophylaxis, andadamantanamino and flumidine have been used for influenza prophylaxis.-Iodo-2'-desoxyuridine has been used for treatment of some cases ofherpes keratitis.

Guanidine and 2-(a-hydroxyphenyl)-benzoimidazole are known to inhibit invitro the multiplication of most picorna viruses, while having no effectat all when applied in vivo. This is due to the quick formation ofmutants stable to the action of these substances.

It is an object of the invention to provide new substances showing highactivity for certain picornaviruses.

SUMMAR Y OFTHE INVENTION In accordance with the invention, there areprovided compounds of the formula wherein R is methyl, ethyl, allyl,propyl, butyl or a benzyl group;

R is methyl, propyl, butyl, benzyl or a cyclohexyl group;

R is sulphur or oxygen;

.X is hydrogen, chlorine or sulphonamide group;

Y is hydrogen or bromine.

DETAILED DESCRIPTION OF THE INVENTION The compounds (I) are preparedunder relatively mild conditions from the reaction of a heterocyclic e ve f fe mul 1 NRl I X (II) with an amine of the formula H N R wherein R RR X and Y are as defined above.

The compounds (I) can be successively applied as virostatics against theaforementioned picornaviruses. Their antiviral properties wereestablished by carrying out in vitro tests on cell cultures and bycarrying out in vivo tests on experimental Coxsackie infections andinfections caused by the FMD virus, by the following methods:

METHOD l The plaque-inhibition test of Herrmann, the compounds beingused in maximum subtoxic concentration.

METHOD II One-step virus growth cycle experiments in which theconcentration of thetested substances equals the maximum subtoxic one.

The investigations with the poliovirus '1, coxsackievirus B1 andechovirus 19 were carried out on cell cultures FL (in some of the casespoliovirus cells KB were used), while calf kidney primary cultures wereused for the FMD virus.

The maximum subtoxic concentration of the compounds can be determined bythe use of graphs which show the multiplication of a given cell culturein the presence of the respective active compound which is followeduntil a stationary phase is reached.

The activity of the compounds was tested in cases of experimentalcoxsackievirus A6, A7, Bl and B3 infections on newborn mice, and incases of F MD virus infections on newborn mice and guinea pigs. In thecases of viral infections on newborn mice the percentage of lethality inthe treated animal groups compared with the placebo group is determined,as well as the alternative response time (BT Link, 1969). When carryingout tests on guinea pigs by inoculating them with FMD virus, the effectshown by the compounds of the present invention is measured on the baseof the percentage of animals in the treated animal groups showinggeneralized infection, compared with the placebo group.

The invention is further illustrated by the following nonlimitiveexamples:

EXAMPLE I N-methyl-N-(2-hydroxy-5-chlorophenyl)-N'- benzylthiourea 1.0g. of N-methyl-S-chlorobenzoxazole-thlone and 1 cc. of benzylamine wereheated at C. After cooling the reaction mixture was dissolved in etherand then consecutively extracted with 6 percent hydrochloric acid and 5percent solution of sodium base. The alkaline extract was thenneutralized, a white crystalline mass falling as precipitate. Yield 1.35g (88 percent of the theoretical). The product was furtherrecrystallized Analysis, Calc. for C H ClN SO: C, 58.81; H, 4.90;

was neutralized and the formed precipitate filtered. After dissolving in10 percent sodium base the product was precipitated with dilutehydroxhloric acid,'the so purified substance weighing 1.85 g. (72percent of the theoretical). The product was further recrystallized 58.'H46'N9.43' Found, C. from alcohol solution. M.p. l35-136C.

' Analysls, Calc. for C H N O C, 71.83; H. 7.04; N,

9.85 EXAMPLES 2 9 Found: c, 71.82; H, 7.15; N, 9.71

- By following the procedures of Example I, substitut- EXAMPLES 1 1 16mg an equimolar amount of the hetero y c Substitution of equimolaramounts of heterocycllc pound (I1) and amine (111) for the N-methyl-S-compound (11) and amine (111) forN-benzylbenzoxazochlorobenzoxazole-thione .and benzylamme, respecloneand propylamlne, respectively m the procedure of tively, antiviralcompounds (1) are produced: Example 10 yields the following results:

Heterocyclic Amine Empirical Allllyhh Ex. Compoundfll) (lll) M.P.(C)formula Found Calc.

2 N-methyl-o-bromobenzylhenzoxazolethione amine 126-128 C, H, BrN OSC,5l.27 C,51.43 N, 8.22 N, 8.00 3N-methyl-o-bromopropylbenzoxazolethione amine 106-107 C H BrN OS N, 8.97N, 9.24 4 N-ethyl-bcnzbenzyl C 7.18 C,67.13 oxazolethione amine 146-147c n w os H, 5.97 H, 6.29 N, 9.63 N, 9.79 5 N-allyl-benzoxbenzyl- C,68.28C 8.45 azolethione amine 142-145 C H N OS H, 6.69 H, 6.08 N, 9.51 N,9.38 6 N-propyl-benzbenzene- 145-146 C, H ,,N OS C 8.26 C,68.00oxazolethione amine H, 6.65 H, 6.66 N, 9.31 N, 9.33 7N-propyl-henzpropyl- C 198 C,61.90 oxazolethione amine 128-129 C H N OSH, 7.29 H, 7.95 N,ll.36 N,1l.ll 8 N-propyl-benzbutyl- C 3.41 C 3.15oxazolethione amine 119-121 C H- N OS H, 8.24 H, 8.27 N,10.85 N,10.52 9N-propyI-henzcyclo- C 6.05 C,65.74 oxazolethione hexyl- H, 8.78 H, 8.21amine 157-158 C H N OS N, 9.64 N, 9.58

Heterocyclic Amine Empirical Analysis Ex. Compound(l1) 1 (111) M.P.(C)Formula Found Calc.

1 1 N-benzyLbenzmethyloxazolone amine 152-153 C,,,H,,,N O., C,70.01(.7031 N,10.83 N,10.93 12 N-benzyl-S- methyl- (1.62.17 C,62.06chloro-benzoxaamine 148-149 C, H C1N. .O. H, 5.96 H, 5.16 zolone N, 9.43N, 9.65 C.64.30 (',64.15 13 N-benzyl-5- propyl- 124 -126 C ,H,,,C1N O H,6.17 H, 5.97 chloroamine N, 8.71 N, 8.80 benzoxazolone 14 N-benzyl-6-propyl- C,56.43 (,5619 bromoamine 129-131 C, H,,,BrN O H, 5.67 H, 5.23henzoxazolone N, 7.43 N, 7.43 15 N-benzylhenzyl- 076.03 C,75.90

benzoxazolone amine 126-127 C H N O 76.00

6.28 16 N-benzyl-S- benzyl- C,69.16 (1,611.85 chloroamine 139 -141 C HCIN O, H, 5.21 H, 5.19

benzoxazolone EXAMPLE 10 N-benzy1-N-(2-hydroxyphenyU-N-propy1urea 2.0 g.of N-benzylbenzoxasolane and 10.0 cc. of percent aqueous solution ofpropylumine were boiled together for 7 hours. After cooling the reactionmixture EXAMPLE 17 Following the test procedures indicated in the table.representative compounds were tested with the results indcated below.

EFFECTS ON PLAQUE FORMATION AND MULTIPLICATION OF PICORNAVIRUSES 0FCOMPOUNDS USED IN MAXIMAL SUBTOXIC CONCENTRATION '7 Virus multiplicationPlaque-formations laque- (one-step virus rowth inhibition test cycletest Diameter PFU/ Percent of PFU/ Poroont of Virus Active compound mm.)meters reduction ml. inhibition r on 1 1X10 68 5 1710 S NHOHaCeHs CH3OQNH,

NC 8 NHCHzCaHs JJHa Polio 1 1 NC S NHCHzCeHn J1aH1 HO 2. 6x10 93. 3

IIIO SNHCHzCoHs CHnCH=CH2 Control sample 4. 0 160, 160 3. 9X10 OH 1. 272, 74 41. 1 3. 4X10 92. 1

IIIC S NHCHzCuHa CHa S OzNHz HO 1. 0 75, 77 38. 7 5. 4X10 87. 5

Coxsackie B1 DIIO S NHCHzCoHa OH 4. 3X10 90. 0

NC 8 NHCHzCoHa Control sample 3.6 128,120 4. 8x10 OH 2. 3 18, 20, 22 39.4 4. 1X10 70. 7

IIIC SNHCHzCoHu CaH1 ECHO 0 1 4. 0x10 11. o

IIIC SNHO iizOeHo CH: Br

Control sample 8 5 82, 33, 84 1. 4X10 NC ONHCHnOo s FMD I OHaCaHs BrControl sample 2. 6X10 In cases of experimental infection in newbornmice caused by coxsackievirus Bl, B3, A6 and A7 (-10 LD virus dose) adaily application of N-methyl-N-(2- hydroxyphenyl-4-sulphonamide)-N-benzylthiourea and N-propyl-N-(2-hydroxyphenyl)-N-benzylthiourea bysubcutaneous administration results in retarding the course of theinfection generally by 2-3 days (BT and in reduction of the number ofdeath cases. ED is 86.6 mg/kg when subcutaneously applied once daily fora period starting 48 hours before viral inoculation and throughout thewhole course of the infection.

In cases of FMD infection on newborn mice application ofN-benzyl-N-(2-hydroxyphenyl)-N'-benzylurea is daily doses of 86.6 mg/kg(acute toxicity LD =2O0.O mg/kg) according to the above said patternprotects from ailing (encephalitic) 83.4-100 percent of the treatedanimals compared with 63.7-90 percent lothality in the placebo group. Incases of infecting guinea pigs with the FMD virus this substance whenadministered in daily doses of 40 mg/kg for a period starting 48 hoursbefore the viral inoculation and ending on t he 5th day after, reducesthe number of disease cases 2 to 3.5 times as compared with the placebogroup (significance level less than 0.01

Investigation of acute toxicity for mice and rats of N-propyl-N-(2-hydroxyphenyl)-N-benzylthiourea showed the following LDvalues (calculated by the Pershin method) for mice-283.5 mg-kg(intraperitoneally) and 733.5 mg-kg (subcutaneously); for rats 2 600.9rug/kg intraperitoneall y) Selectivity index (LD50/ED50) iS Whenpregnant female rats are intraporitoneally treated with N-propyl-N-(2-hydroxyphenyl )-N benzylthiourea (daily doses of 50 mg/kg) noteratogenic effects are observed.

What is claimed is:

1. N-methyl-N-(2-hydroxy-5-chlorophenyl)-N- benzylthiourea.

2. N-methyl-N-( 2-hydroxy-6-bromophenyl)-N propylthiourea.

1. N-METHYL-N-(2-HYDROXY-5-CHLOROPHENYL)-N''BENZYLTHIOUREA. 2.N-methyl-N-(2-hydroxy-6-bromophenyl)-N''-propylthiourea.